Posttranslational modification of proteins is often necessary for proper protein folding and function. A common protein modification is the addition of oligosaccharides (glycans) to nascent polypeptides in the endoplasmic reticulum to form glycoproteins, a process known as glycosylation. N-glycosylation is of particular importance in the production of recombinant proteins used for therapeutic purposes. Because standard prokaryotic expression systems lack the proper machinery necessary for such modifications, alternative expression systems have to be used in production of these therapeutic proteins. Yeast and fungi are attractive options for expressing proteins as they can be easily grown at a large scale in simple media, which allows low production costs. Moreover, tools are available to manipulate the relatively simple genetic makeup of yeast and fungal cells as well as more complex eukaryotic cells such as mammalian or insect cells (De Pourcq et al., Appl Microbiol Biotechnol, 87(5):1617-31).
Fungal cells and mammalian cells share common steps in the early stages of glycosylation that result in the formation of mannose(8)N-acetylglucosamine(2) (Man8GlcNAc2). However, significant differences exist in the later stages of the process. For example, in yeast, additional mannose subunits are added to Man8GlcNAc2 by mannosyltransferases and mannan polymerases to yield high-mannose type N-glycans. In contrast, mannose sugars are removed from the human Man8GlcNAc2 to yield Man5GlcNAc2, followed by three sequential reactions involving the enzymes N-acetylglucosaminyltransferase I (GnTI), mannosidase II (Mns II), and N-acetylglucosaminyltransferase II (GnTII), to convert Man5GlcNAc2 into GlcNAc2Man3GlcNAc2.
The differences between the glycosylation process in mammalian and fungal cells pose a challenge to the expression of glycosylated mammalian proteins in fungal cells since glycoproteins with high-mannose type N-glycans are not suitable for therapeutic use in humans (De Pourcq et al., 2010; Wildt and Gerngross, Nature Reviews Microbiology, 3: 119-128). Consequently, studies have been conducted to re-engineer the glycosylation pathways in yeast and fungal species to enable them to express recombinant human proteins. The general approach in glycoengineering of yeast or fungal cells has been to disrupt endogenous genes that are involved in formation of high-mannose type N-glycans. These gene disruptions can be combined with over-expression of endogenous mannosidases and/or glycosyltransferases and glycosidases from different species (Chiba et al., 1998, J Biol Chem 273: 26298-304; Kainz et al., 2008, Appl Environ Microbiol 74: 1076-86; Maras et al., 1997, Euro J Biochem 249: 701-07; Maras et al., 1999, Febs Letters 452: 365-70; Hamilton et al., 2003, Science 301: 1244-6; De Pourcq et al., 2010). However, the production of glycosylated mammalian proteins in non-mammalian cells still requires complicated and time-consuming genetic engineering and can be inefficient at producing a desired glycoprotein.
Thus, a need remains in the art for a simpler and more efficient system to express complex N-glycans in non-mammalian cells.